To capitalize on the enormous potential afforded by the ability to perform targeted gene disruption experiments in mammalian cells we need more information concerning the homologous recombination process. To this end we propose to develop a convenient and reliable assay with the ability to detect and quantitate mammalian cell transformants in which targeted integration via homologous recombination has occurred. The assay is based on the use of thymidine kinase (TK) deficient cell lines with highly amplified copies of functional adenosine deaminase (ADA) structural genes which serve as the target for homologous integration. In these cells ADA levels have increased over 10,000-fold and account for over 75% of the soluble protein. The increase in enzyme activity is accompanied by a corresponding increase in the number of ADA genes, a correlation suggesting that all of the amplified ADA genes account for over 5% of the genome in these cells, thus providing an extremely large number of targets for homologous recombination with incoming DNA molecules. A cloned fragment from the ADA structural gene will be placed in front of a promoterless thymidine kinase (TK) minigene in such a way that homologous recombination into any one of the 20,000 ADA structural genes will enable TK activity to be expressed as an ADA/TK fusion protein. TK+ transformants will be identified by their ability to grow in HAT medium. Control experiments using cell lines without amplified ADA genes and constructs with and without the ADA structural gene fragment in front of the TK minigene will enable us to decide if homologous recombination is occurring at a significant frequency. If experiments are successful then these cell lines and molecular constructs will constitute a gene transfer and expression assay that will allow easy detection and quantitation of homologous recombination in mammalian cells. We will use this assay with recipient cells having fewer and fewer ADA genes to determine the relationship between the number of target sites and the frequency of targeted integration. This assay should also enable other investigators to determine the effects of various experimental parameters on the frequency of targeted integration.